The symbol originates with the medieval Danse Macabre symbolism. By the 15th century, the symbol had developed into its familiar form. It is used in military flags or insignia, expressing the recklessness or ferocity of the unit displaying it, since at least the 15th century and becomes associated with piracy in particular in the 18th. It came to be used specifically to mark the entrances of Spanish cemeteries. The use of the symbol to mark poison dates to the 19th century.

In 1829, New York State required the labeling of all containers of poisonous substances. The skull and crossbones symbol appears to have been used for that purpose since the 1850s. Previously a variety of motifs had been used, including the Danish "+ + +" and drawings of skeletons.

In the 1870s poison manufacturers around the world began using bright cobalt bottles with a variety of raised bumps and designs (to enable easy recognition in the dark) to indicate poison, but by the 1880s the skull and cross bones had become ubiquitous, and the brightly coloured bottles lost their association.

Typical cartoon pirate sporting the skull-and-crossbones on his hat

As the skull-and-crossbones symbol has also entered popular culture in the context of piracy, and since cartoonish pirates have become popular characters with children, there have been concerns that the "poison" symbol might have the effect of attracting the curiosity of small children familiar with "pirates" as depicted as a toy or play theme. For this reason, in the United States there has been a proposal to replace the skull and crossbones by the "Mr. Yuk" symbol. However, Mr. Yuk and his graphic rendering are registered trademarks and service marks of his creator, the Children's Hospital of Pittsburgh of UPMC, and the rendering itself is additionally protected by copyright. This means that the name and graphic image cannot be used without a license from the owner—unlike the Skull and crossbones, which is in the public domain.